Genome Organization and Adaptive Potential of Archetypal Organophosphate Degrading Sphingobium fuliginis ATCC 27551.

Sphingobium fuliginis ATCC 27551, previously classified as Flavobacterium sp. ATCC 27551, degrades neurotoxic organophosphate insecticides and nerve agents through the activity of a membrane-associated organophosphate hydrolase. This study was designed to determine the complete genome sequence of S. fuliginis ATCC 27551 to unravel its degradative potential and adaptability to harsh environments. The 5,414,624 bp genome with a GC content of 64.4% is distributed between two chromosomes and four plasmids and encodes 5,557 proteins. Of the four plasmids, designated as pSF1, pSF2, pSF3, and pSF4, only two (pSF1 and pSF2) are self-transmissible and contained the complete genetic repertoire for a T4SS. The other two plasmids (pSF3 and pSF4) are mobilizable and both showed the presence of an oriT and relaxase-encoding sequences. The sequence of plasmid pSF3 coincided with the previously determined sequence of pPDL2 and included an opd gene encoding organophosphate hydrolase as a part of the mobile element. About 15,455 orthologous clusters were identified from among the cumulatively annotated genes of 49 Sphingobium species. Phylogenetic analysis done using the core genome consisting of 802 orthologous clusters revealed a close relationship between S. fuliginis ATCC 27551 and bacteria capable of degradation of polyaromatic hydrocarbon compounds. Genes coding for transposases, efflux pumps conferring resistance to heavy metals, and TonR-type outer membrane receptors are selectively enriched in the genome of S. fuliginis ATCC 27551 and appear to contribute to the adaptive potential of the organism to challenging and harsh environments.

Topological analysis of the lipoprotein organophosphate hydrolase from Sphingopyxis wildii reveals a periplasmic localisation.

Organophosphate hydrolase (OPH) is a membrane-associated lipoprotein. It translocates across the inner membrane via the twin-arginine transport pathway and remains anchored to the periplasmic face of the inner membrane through a diacylglycerol moiety linked to the invariant cysteine residue found at the junction of a SpaseII cleavage site. Due to the existence of a transmembrane helix at the C-terminus of the mature OPH, an inner-membrane topology was predicted suggesting the C-terminus of OPH is cytoplasmic. The predicted topology was validated by generating OPH variants either fused in-frame with ß-lactamase or with unique cysteine residues. Sphingopyxis wildii cells expressing OPH variants with Bla fused at the N-terminal, C-terminal or central regions all grew in the presence of ampicillin. Supporting the ß-lactamase reporter assay, the OPH variants having unique cysteine residues at different strategic locations were accessible to the otherwise membrane-impermeant PEG-Mal (methoxypolyethylene glycol maleimide) revealing that, with the exception of the lipoprotein anchor, the entire OPH is in the periplasmic space.

Genome-Guided Insights Reveal Organophosphate-Degrading Brevundimonas diminuta as Sphingopyxis wildii and Define Its Versatile Metabolic Capabilities and Environmental Adaptations.

The complete genome sequence of Brevundimonas diminuta represented a chromosome (∼4.15 Mb) and two plasmids (pCMS1 and pCMS2) with sizes of 65,908 and 30,654 bp, respectively. The sequence of the genome showed no significant similarity with the known bacterial genome sequences, instead showed weak similarity with the members of different genera of family, Sphingomonadaceae. Contradicting existing taxonomic position, the core genome-guided phylogenetic tree placed B. diminuta in the genus Sphingopyxis and showed sufficient genome-to-genome distance warranting a new species name. Reflecting the strains ability to grow in harsh environments, the genome-contained genetic repertoire required for mineralization of several recalcitrant man-made aromatic compounds.

Organophosphate Hydrolase Is a Lipoprotein and Interacts with Pi-specific Transport System to Facilitate Growth of Brevundimonas diminuta Using OP Insecticide as Source of Phosphate.

Organophosphate hydrolase (OPH), encoded by the organophosphate degradation (opd) island, hydrolyzes the triester bond found in a variety of organophosphate insecticides and nerve agents. OPH is targeted to the inner membrane ofBrevundimonas diminutain a pre-folded conformation by thetwinargininetransport (Tat) pathway. The OPH signal peptide contains an invariant cysteine residue at the junction of the signal peptidase (Spase) cleavage site along with a well conserved lipobox motif. Treatment of cells producing native OPH with the signal peptidase II inhibitor globomycin resulted in accumulation of most of the pre-OPH in the cytoplasm with negligible processed OPH detected in the membrane. Substitution of the conserved lipobox cysteine to serine resulted in release of OPH into the periplasm, confirming that OPH is a lipoprotein. Analysis of purified OPH revealed that it was modified with the fatty acids palmitate and stearate. Membrane-bound OPH was shown to interact with the outer membrane efflux protein TolC and with PstS, the periplasmic component of the ABC transporter complex (PstSACB) involved in phosphate transport. Interaction of OPH with PstS appears to facilitate transport of Pigenerated from organophosphates due to the combined action of OPH and periplasmically located phosphatases. Consistent with this model,opdnull mutants ofB. diminutafailed to grow using the organophosphate insecticide methyl parathion as sole source of phosphate.

The Organophosphate Degradation (opd) Island-borne Esterase-induced Metabolic Diversion in Escherichia coli and Its Influence on p-Nitrophenol Degradation.

In previous studies of the organophosphate degradation gene cluster, we showed that expression of an open reading frame (orf306) present within the cluster in Escherichia coli allowed growth on p-nitrophenol (PNP) as sole carbon source. We have now shown that expression of orf306 in E. coli causes a dramatic up-regulation in genes coding for alternative carbon catabolism. The propionate, glyoxylate, and methylcitrate cycle pathway-specific enzymes are up-regulated along with hca (phenylpropionate) and mhp (hydroxyphenylpropionate) degradation operons. These hca and mhp operons play a key role in degradation of PNP, enabling E. coli to grow using it as sole carbon source. Supporting growth experiments, PNP degradation products entered central metabolic pathways and were incorporated into the carbon backbone. The protein and RNA samples isolated from E. coli (pSDP10) cells grown in (14)C-labeled PNP indicated incorporation of (14)C carbon, suggesting Orf306-dependent assimilation of PNP in E. coli cells.

OxyR-dependent expression of a novel glutathione S-transferase (Abgst01) gene in Acinetobacter baumannii DS002 and its role in biotransformation of organophosphate insecticides.

While screening a genomic library of Acinetobacter baumannii DS002 isolated from organophosphate (OP)-polluted soils, nine ORFs were identified coding for glutathione S-transferase (GST)-like proteins. These GSTs (AbGST01-AbGST09) are phylogenetically related to a number of well-characterized GST classes found in taxonomically diverse groups of organisms. Interestingly, expression of Abgst01 (GenBank accession no. KF151191) was upregulated when the bacterium was grown in the presence of an OP insecticide, methyl parathion (MeP). The gene product, AbGST01, dealkylated MeP to desMeP. An OxyR-binding motif was identified directly upstream of Abgst01. An Abgst-lacZ gene fusion lacking the OxyR-binding site showed a drastic reduction in promoter activity. Very low ß-galactosidase activity levels were observed when the Abgst-lacZ fusion was mobilized into an oxyR (GenBank accession no. KF151190) null mutant of A. baumannii DS002, confirming the important role of OxyR. The OxyR-binding sites are not found upstream of other Abgst (Abgst02-Abgst09) genes. However, they contained consensus sequence motifs that can serve as possible target sites for certain well-characterized transcription factors. In support of this observation, the Abgst genes responded differentially to different oxidative stress inducers. The Abgst genes identified in A. baumannii DS002 are found to be conserved highly among all known genome sequences of A. baumannii strains. The versatile ecological adaptability of A. baumannii strains is apparent if sequence conservation is seen together with their involvement in detoxification processes.

Multiple mechanisms contribute to lateral transfer of an organophosphate degradation (opd) island in Sphingobium fuliginis ATCC 27551.

The complete sequence of pPDL2 (37,317 bp), an indigenous plasmid of Sphingobium fuliginis ATCC 27551 that encodes genes for organophosphate degradation (opd), revealed the existence of a site-specific integrase (int) gene with an attachment site attP, typically seen in integrative mobilizable elements (IME). In agreement with this sequence information, site-specific recombination was observed between pPDL2 and an artificial plasmid having a temperature-sensitive replicon and a cloned attB site at the 3' end of the seryl tRNA gene of Sphingobium japonicum. The opd gene cluster on pPDL2 was found to be part of an active catabolic transposon with mobile elements y4qE and Tn3 at its flanking ends. Besides the previously reported opd cluster, this transposon contains genes coding for protocatechuate dioxygenase and for two transport proteins from the major facilitator family that are predicted to be involved in transport and metabolism of aromatic compounds. A pPDL2 derivative, pPDL2-K, was horizontally transferred into Escherichia coli and Acinetobacter strains, suggesting that the oriT identified in pPDL2 is functional. A well-defined replicative origin (oriV), repA was identified along with a plasmid addiction module relB/relE that would support stable maintenance of pPDL2 in Sphingobium fuliginis ATCC 27551. However, if pPDL2 is laterally transferred into hosts that do not support its replication, the opd cluster appears to integrate into the host chromosome, either through transposition or through site-specific integration. The data presented in this study help to explain the existence of identical opd genes among soil bacteria.

Organophosphate hydrolase in Brevundimonas diminuta is targeted to the periplasmic face of the inner membrane by the twin arginine translocation pathway.

A twin arginine translocation (Tat) motif, involved in transport of folded proteins across the inner membrane, was identified in the signal peptide of the membrane-associated organophosphate hydrolase (OPH) of Brevundimonas diminuta. Expression of the precursor form of OPH carrying a C-terminal His tag in an opd-negative background and subsequent immunoblotting with anti-His antibodies showed that only the mature form of OPH associated with the membrane and that the precursor form of OPH was entirely found in the cytoplasm. When OPH was expressed without the signal peptide, most of it remained in the cytoplasm, where it was apparently correctly folded and showed activity comparable to that of the membrane-associated OPH encoded by the wild-type opd gene. Amino acid substitutions in the invariant arginine residues of the Tat signal peptide affected both the processing and localization of OPH, confirming a critical role for the Tat system in membrane targeting of OPH in B. diminuta. The localization of OPH to the periplasmic face of the inner membrane in B. diminuta was demonstrated by proteinase K treatment of spheroplasts and also by fluorescence-activated cell sorting analysis of cells expressing OPH-green fluorescent protein fusions with and without an SsrA tag that targets cytoplasmic proteins to the ClpXP protease.